Antigen-binding polypeptides such as single chain variable fragments (scFv) comprise of the variable domains of the light (VL) and heavy (VH) chain of a corresponding full-length antibody. A similar architecture has also been applied to the structurally similar T cell receptors (scTv) as well as scFab-fragments. In such constructs, both chains are normally connected by a linker which is flexible and does not show any tendency to interfere with folding of the individual immunoglobulin domains. In many cases, these linkers contain assemblies or variations of (Gly4Ser) (SEQ ID NO: 136) repeats, inspired by the unstructured linkers connecting the domains of filamentous bacteriophage minor coat protein III.
ScFv antibody fragments are widely used in a variety of applications, such as for research, diagnostic purposes and even as therapeutics. Immunotoxins, which are used for cancer therapy, are often based on a single chain fragment fused to a bacterial toxin to mediate targeted killing. Another approach is based on bispecific antibodies (BiTEs, bispecific T cell engagers) which activate and redirect cytotoxic T cells against cancer cells. CAR (chimeric antigen receptor)-T cell therapy also relies on scFvs specific for malignant cells. Essential for all of these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes. These properties would also be very useful for the purification of biomaterials, in particular proteins, vaccines or cells. However, the usually very high affinity of antibodies requires harsh elution conditions, which typically impairs folding, integrity or viability of the eluted materials. Therefore, antibodies which retain their excellent specificity while being adjustable in respect of their affinity without requiring harsh conditions for this adjustment would be advantageous for protein purification, cell separation and cell analysis. Even the introduction of an affinity-adjustable antibody for therapy may be envisioned, for example as an additional safety mechanism in CAR-T cell therapy.
Kobatake, E. et al. (2012, Biotechnol Lett 34, 1019-23) disclose an affinity changeable antibody in response to calcium. The system is based on a fusion-peptide comprising scFv, wild-type (WT) calmodulin, and a calmodulin-binding peptide. The switch is generated by the addition of calcium to the system. One disadvantage is that the solution must be calcium-free before the intended switch.
Meister, G. E. & Joshi, N. S. (2013, Chembiochem 14, 1460-7) disclose a switchable enzyme which bases on the interaction of WT-calmodulin and soluble M13 peptide. In the peptide-bound form the enzyme exhibits an up to 120 times higher catalytic activity compared to the inactive (no peptide bound) state.
WO2002014371A1 discloses Fv constructs having an affinity that can be influenced for a substance to be linked, wherein the Fv constructs have peptides linked to the variable regions and containing binding sites for effector molecules. The effector molecules are ions or antibodies.
Guntas, G. et al. (2004, Chem Biol 11, 1483-7) and WO2003078575A2 disclose the creation of a molecular switch of the enzyme TEM1 β-lactamase by circularly permutating the gene encoding the enzyme TEM1 β-lactamase and subsequently inserting it into the gene encoding E. coli maltose binding protein which functions as the linker.
WO2005/072392A2 discloses molecular switches, for example with switching activity greater than previously demonstrated, or with altered ligand recognition and binding, and methods of making these molecules involving circular permutation of nucleic acid or amino acid sequences. Molecular switches have been created by recombining non-homologous genes in vitro and subjecting the genes to evolutionary pressure using combinatorial techniques. The approach is envisioned as “rolling” two proteins across each other's surfaces and fusing them at points where their surfaces meet. The approach allows for recombination and testing of maximal numbers of geometric configurations between the two domains. Libraries comprising vast numbers of such fused molecules are provided from which molecular switches with optimal characteristics can be isolated.
Megeed, Z. et al. (2006, Biomacromolecules 7, 999-1004) disclose a fusion peptide of scFv with elastin as linker resulting in a temperature dependent affinity of the antigen binding domain to the antigen.
Miyawaki, A. et al. (1997, Nature 388, 882-887) disclose a polypeptide comprising a fluorescent protein, wherein its domains are linked by a calmodulin-M13-peptide. Baird et al. (1999, PNAS 96: 11241-11246) showed that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still provide fluorescence. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced several-fold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca2+ in single mammalian cells.
Nagai et al. (2001, PNAS 98:3197-3202) showed by using a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini that they could visualize Ca2+-dependent protein-protein interactions in living cells by fluorescence readouts. The cpGFP was fused to calmodulin and its ligand derived peptide, M13. The chimeric protein was fluorescent and its spectral properties changed reversibly with the amount of Ca2+.
Calmodulin (CaM) undergoes large conformational changes, depending on the presence of calcium and calmodulin-binding peptides (CBP). In a calcium- and peptide-unbound form, it adopts a closed conformation (Kuboniwa, H. et al., 1995, Nat Struct Biol 2, 768-776). The distance between the N- and C-terminus is at its highest in the calcium-bound, open form (Chattopadhyaya, R. et al., 1992, J Mol Biol 228, 1177-1192), whereas the termini approach each other when calmodulin binds to a ligand, or a suitable fragment thereof, like peptide M13 (Ikura, M. et al., 1992, Science 256, 632-638).
Montigiani et al. (1996, J. Mol. Biol. 258:6-13) and Hultschig et al. (2004, J. Mol. Biol. 343:559-568) identified high affinity mutants of the CaM binding peptide “M13” which is derived from the rabbit myosin light chain kinase.
There is a need in the art for an alternative or improved composition and/or method for affecting the binding of antigen-binding polypeptides to their respective antigens.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.